Process of producing mixture of ptc, proconvertin and prothrombin



Sept. 12, 1961 A. H: FowELl. 2,999,791

PROCESS OF PRQDUCING MIXTURE OF PTC, PRocoNvERTIN AND PRoTHRoMBIN Filed Aug. 6, l1957 2 Sheets-Sheet 1 a DAYS v INVENTOR. 2 ALFRED /7C Fon/ELL Sept. 12, 1961 Filed Aug. 6, 1957 A. H. FOWELL PROCESS OF PRODUCING MIXTURE OF' PTC,

PROCONVERTIN AND PROTHROMBIN 2 Sheets-Sheet 2 IN VEN TOR. ALF/aro /f Fon/ELL Irfan/5K5 nited States Patent C f Filed Aug. 6, 1957, Ser. No. 676,557 3 Claims. (Cl. 167-74)v This invention relates to blood coagulation components and in general has for its object the provision of a purified composition of blood coagulation components and to. al method for producing such composition.

The blood coagulation mechanism is extremely complicated and involves the interaction of at least 19 or 20 different protein substances, including plasma thromboplastin component, proconvertin, and prothrombin. Although the coagulation mechanism is conveniently diagrammed in Albrittons Standard Values in Blood, pages l1 through l5 (W. B. Saunders Co., 1952), Albrittons diagrams do not show the role played by plasma thromboplastin component, for this component has been discovered only recently.

Plasma thromboplastin component is a component which is missing from the blood of all patients aticted with hemophilia B, and is essential for the formation of plasma thromboplastin. The other thromboplastin precursors are thromboplastinogen (missing from the blood of hemophilia A patients) and a platelet factor. Proeonvertin is a component which is essential for the rapid conversion of prothrombin to thrombin. Prothrombin is the actual precursor of thrombin.

Because of the primitive state of knowledge concerning blood coagulation, confusion may result from variations in nomenclature, and in attempting to. avoid this it should be noted that plasma thromboplastin component is also known as Plasma Thromboplastin Factor-B, and Christmas Factor, and that proconvertin is also known as Stable Component, Factor VII (Kohler), and Serum Prothrombin Conversion Accelerator Precursor.

There are estimated to be 60,000 cases of congenital hemophilia in the United States. Of these, approximate.- ly 9000 yare cases of hemophilia B, the blood of such patients being either totally devoid of plasma thromboplastin component r seriously deficient in plasma thromboplastin component. The disease therefore exists in varying degrees of severity, requiring therapy anywhere from every three Weeks up to once or twice a year. The completely deficient cases require replacementA therapy once every three weeks; the partially deticient cases, which are the majority of cases, require therapy only when bleeding episodes occur, which may be as seldom as once a year. The bleeding episodes in congenital, partially-deficient cases are generally caused by a temporarily acquired severe deficiency rather than by injury alone. Intravenous injection of approximately 800 ml. of fresh plasma, or yan equivalent amount (about 1200 ml.) of fresh blood, temporarily corrects the defect of a completely deficient subject. The beneficial effect lasts for about three weeks, although the coagulation defeet, asV measured by in vitro tests on the patients blood appears normal for only three or four days. Such ther- -apy with fresh plasma or fresh -blood is effective but it has two important drawbacks: (l) it requires ready Vavailability of a large amount of fresh blood and requires hospitalization for the administration of the blood; 2) a great many of the patients eventually become sensitized to repeated blood infusions and ultimately encounterv fatal transfusion reactions.

.'I'here are estimated to be about 400 patients with con- Y 'genital proconvextindeficiency still. living in the United States. These are serious cases, and the patients seldom survive beyond childhood. Intravenous injection of approximately 300 ml. of fresh plasma, or an equivalent amount (about 500 ml.) of fresh blood, temporarily corrects the proconvertin deficiency in a child. Repeated therapy of proconvertin deficiency with plasma or blood is subject to the same costs and hazards as pointed out above in connection with plasma thromboplastin component deficiency. Proconvertin deficiency is also temporarily acquired during some liver infections, during some transfusion reactions, and hom overdosage with dicum-arol, and if prompt treatment with an available therapeutic agent were possible, it would be life-savings.

in many cases.

At the present time I do not know of anyl incidence of congenital prothrombin deficiency. However, temporary prothrombin deficiencies are acquired in the same manner as the temporary proconvertin deficiencies. The infusion of fresh blood or plasmav into patients for some unknown reason does not produce any measurable increase in lthe prothrombin blood level.

More specifically, one of the objects of this invention is the provision of a synergistic mixture of human blood coagulation components, including plasma thromboplastin component, proconvertin, and prothrombin, and which may be used far more effectively than fresh blood or plasma. i

Another object of this invention is the provision of a process for preparing said mixture from human blood plasma wherein said coagulation components are selectively adsorbed on an adsorbent such as barium sulfate; the adsorbent is separated from the plasma; the coagulants are eluted from the adsorbent with sodium citrate; and finally, the coagulants are precipitated from the sodium citrate solution.

In the drawings hereof:

FG. l includes a pair of curves illustrating the effect on a hemophilia B patient of the administration of various amounts of plasma as compared with the administration of various amounts of the product of this invention.

FIG. 2 includes a pair of curves illustrating the elect on Ithe prothrombin consumption and prothrombin plus proconvertin consumption of a hemophilia B patient following the injection of 1120 rnl. of fresh plasma and following the injection of 150 mg. of my product (No. l 141).

FIG. 3 includes three curves illustrating the comparative effects on the clotting time in a hemophilia B patient resulting from the administration of 560 ml. of fresh plasma, 1120 ml. of fresh plasma, and 150 mg. of my product (No. 1141). i

appended hereto Iand made a part Details of process By way of illustration, the processabove referred to can be carried out as follows. j

(l) Collect blood into an anticoagulant solution containing 750 mg. disodium ethylenediamne tetra-acetate in 20 ml. water for every 500 ml. blood. The cellular elements are separated and discarded.

(2) Adjust 100 liters of the plasma to pI-Il 6.0 and add 4 kg. of finely powdered barium sulfate. After stirring for approximately 30 minutes, separate the plasma fromthe barium sulfate. The plasma can then be fractionated into its other clinically useful proteins by conventional means. i

(3) Wash the barium sulfate with l001ters of 0.9 percent sodium chloride. n

(4) Elute a fraction of the adsorbed proteins from the barium sulfate using about liters of 4 percent sodium citrate adjusted to pH 7.5. Discard the barium sulfate. L

Vpurification by the removal of some impurities. ,omits step No. 5" and goes directly to step No. ,6, the

l (5) Adjust the eluate to pH 7.0, 15 percent ethanol, v-5" C., and discard the precipitate which forms.

(6) Adjust the supernatant solution to pI-I 3.2, l5 percent ethanol, 5 C., and collect the precipitate which forms.

b (7) Lyophilize the precipitate. The vresultant dry weight is about 20 grams.

(8) Dissolve the precipitate in about 4.0 liters of aqueous diluent and remove and discard insoluble impurities.

l (9) Sterilize the solution by filtration through a baclteria-excluding filter, and asceptically lill 40 ml. quantities into sterile vials. Q V(l) Lyophilize the content of each vial. The con- -tents ofeach vial should be redissolved in sterile water liust prior to'use. The coagulation components are obtained in an over-all yield of 65 percent or better. The above process recites the details which I prefer at this time. However, many variations can be made Without departing from the general principles of the process. I have tried some variations, and the resultant products were subjected to clinical test in human subiects. This ,type of human testing is so diiicult and so hazardous to the patient, since the patient .must be allowed to reach an incoagulable state prior to thetest, that it is impracltical to test the effects of all reasonable variations in process. Some possible variations have been established, however.

For example, instead of the disodium ethylenediamine tetra-acetate anticoagulant one can substitute a sodium 'oxalate' anticoagulant, 4or during the blood collection the blood can be treated immediately with a calcium-binding 4ion exchange resin such as Dcwex 50, a sulfonic acid styrene type resin manufactured by The Dow Chemical Company. The conventional Anticoagulant Sodium Ci- Itrate Solution, U.S.P., or Anticoagulant Acid Citrate Dex- .trose Solution, U.S.P.,fcannot vbe employed as anticoagulants if one wishes to obtain puritiedplasrna thromboplastin component by my process. If a citrate anticoagulant is used, my iinal product contains the usual amounts of prothrombin and proconvertin but is almost .totally lacking in` invivo plasma thromboplastin component activity. This has been demonstrated repeatedly.

The pH `at step No. 2 can be varied considerably, at least between pH 5.4 and 7.2, but the highest purity along with adequate yield of plasma thromboplastin component and proconvertin is obtained when this step is conducted :near pH 6.0.

. Other adsorbents can be substituted for the barium sulfate. For example, aluminum hydroxide or asbestos can lbe used. Larger or smaller amounts of adsorbents can housed. The use of appreciably smaller amounts of advthe elution. If the pH at step No. 4 is too high, the

coagulation components are gradually destroyed. If the pH is too low, elution is slow and an excessive volume of eluting fluid is required. With these limitations in vrnind, the coagulation components can be eluted to a useful degree at pH values between 6.5 and 8.0.

It effects a higher degree vot If one Step No, is optional.

final product is useful clinically but is of-somewhat lower purity It the pH of step No. 6 is below 3.0, there is a .progressive loss otprothrornbin activity; As the pH is gincreased aboveabout 4;5; there is a progressive loss of yield of plasma thromboplastin component and procenvertin. Although prothromb' can be recovered quantitatively up to pH 5.6, a pH in the region of 3.2 is preferred. The ethanol concentration at step No. 6 can be increased of course, but nothing is gained by doing so. As water-miscible volatile protein precipitants in steps 5 and 6, only eth'anol,`methanol andisopropanol have been tried; all three were satisfactory. Undoubtedly certain other lower alcohols and lower ketones will be satisfactory, but I cannot speciiically predict their utility at this time. l

Step No. 7 is optional. However, following re-solution, a small amount of impurity precipitates and it is desirable to remove this prior to the sterilizing filtration.

The aqueous diluent in step No. 8 can be anything which prevents the adsorption of the coagulation components on the bacteria-excluding filter. I have used successfully such aqueous diluents as a 2.5 percent solution of serum albumin, ldiluted barium sulfate-adsorbed blood serum, and (lmolar glycine.

The step of lyophilization inthe nal containers is also optional. The iinal product is stableas a frozen solution as Well as stable in the dry state. It gradually deteriorates with time when in solution, and I prefer to distribute the product in the dry or frozen state.

Character of product At all events, the product resulting from the process above described consists of a small amount of sterile, fdry powder including a mixture of highly purified plasma thromboplastin component, proconvertin, and lprothrombin. mg. of the product when dissolved in a small quantity of sterile water contains the plasma thromboplastin component, proconvertin, and prothrombin originally present in 800 ml. of fresh plasma or 1200 ml. of fresh blood. The product contains less than 0.3 percent of the total plasma proteins.

Testing limitations There is as yet no reliable method for estimating the potency of the plasma thromboplastin component in vitro. Prothrombin or proconvertin or prothrombin plus proconvertin can be assayed reliably in vitro, but for assessing the potency of a preparation of plasma thromboplastin component it is necessary to rely on the actual intravenous administration of the preparation into a completely-deicient patient and then follow the patients clotting time, percent prothrombin consumption, and thromboplastin generation for two weeks. .The proconvertin content can be Yassayed by the method of Owren and Aas, Scand. I. Clin. Lab. Invest. 3,V 201' (1951). The prothrombin content can be assayed by the method of Kiort, Rapaport, and Ow'ren, I. Lab. Clin. Med. 46, 86, (l955). The combined procouvertin plus prothrombin can be assayed by the method of Ware and Straguell, Am. l. Clin, Pathol. 22, 791 (1952). In spite of optimistic reports in the literature which have never been verified clinically, the potency of purified plasma thromboplastin component as assayed in vitro by either the conventional Prothrombin Consumption Test or the conventional Thromboplastin Generation Test bears no relationship'to the clinical effectiveness of the puritied plasma thromboplastin component in human subjects.

Clinical results In patients congenitally alicted with hemophilia B and completely deficient in plasma thromboplastin component, the intravenous injection of a solution of l5() mg. of my product corrected their coagulation defect for a period of 14 days and maintained the patients symptomfree for varying periods in excess of three weeks.

For example, FIG. 1 compares the immediate eliect on the clotting time of a hemophilia B patient following the injection of varying amounts of fresh plasma and following the injectionof varying amounts of a solution of my 'product (No. 1,141), j Each injection and measurement was made following an interval of at least two Weeks, during which period no treatment was given.

FIG. 2 compares the prolonged effect on prothrombin consumption of a hemophilia B patient following the injection of 1120 ml. of fresh plasma and following the injection of a solution of 150 mg. of product No. 1141. In the prothrombin consumption test, the lower the percent prothrombin, the more effective is the agent in converting prothrombin to thrombin in vivo.

FIG. 3 compares the prolonged effect on clotting time in a hemophilia B patient following the injection of 560 ml. of ACD fresh plasma, 1120 ml. of ACD fresh plasma, and a solution of 150 mg. of product No. 1141.

As the above tests demonstrate, l0 ml. of my product is as effective as approximately 1000 ml. of fresh plasma. Of even greater importance, patients who have become sensitized to whole plasma and who show anaphylactoid reactions to the injection of plasma tolerate the injection of my purified coagulation components with no reactions whatsoever. Naturally, tests comparing my product with plasma cannot be made on these patients.

By various means, I have prepared purified plasma thromboplastin component free of prothrombin. The in vivo administration of prothrombin-free plasma thromboplastin component has not fully beneted hemophilia B patients, nor does the administration of prothrombin alone benefit hemophilia B patients. There is no ready explanation for this phenomenon. When my product containing the three purified coagulation components together is administrated to patients, the plasma thromboplastin component potency in vivo is not related to its prothrombin content or to its proconvertin content but appears to be related to its plasma thromboplastin component content plus its prothrombin content. The effect of injection of the product on the in vivo prothrombin plus proconvertin level is far in excess of that predictable from the amount of these factors injected. I am unable to explain this discrepancy.

In proconvertin deficient patients, my product has been found to be just as effective in remedying the coagulation defect as in plasma thromboplastin component decient or hemophilia B patients. The administration of over 1000 ml. of fresh plasma to such patients does not significantly alter their blood prothrombin level, or proconvertin level, or their combined prothrombin plus proconvertin level, but does temporarily remedy their coagulation defect. However, the administration of my product containing the admixed three coagulation cornponents results in an immediate and prolonged rise in both the proconvertin level and the prothrombin plus proconvertin level.

In both plasma thromboplastin component decient patients and proconvertin deficient patients the administration of fresh plasma does not measurably increase the in vivo plasma prothrombin level. However, the administration of my purified product does increase the in vivo prothrombin level from 60 percent of normal up to 100 percent of normal. Furthermore, the co-administration of prothrombin appears necessary for the plasma thromboplastin component to be fully effective clinically.

Summary The process herein described effects the simultaneous recovery and purification of three inter-related coagulation components. Of great importance is the degree of concentration and purification which is achieved. On a protein basis, the coagulation components are concentrated 400-fold. yIn other Words, 99.75 percent of the total plasma protein is removed and is available for the preparation of other known, clinically useful plasma fractions. The antihemophilia A factor is spared and is available for concentration and purification whenever a suitable process is discovered. The high degree of purity in which the three coagulation components is obtained is also of paramount importance clinically. The removal of 99.75 percent of unneeded plasma protein renders the product suitable for the treatment of the congenital coagulation defects in patients who have become sensitized to Whole plasma, or who have become refractory to treatment with whole plasma, and thus offers these patients a means of renewing maintenance therapy.

The concentration in volume is also of great importance clinically. Aside from the obvious convenience, economy, and safety of a purified concentrate, it is very easy to achieve an effective in vivo blood level of the coagulation components by injection of 10 ml. in a few minutes, whereas it is very difficult to achieve an effective in vivo blood level by injecting 1000 ml. of plasma over a period of 4 to 5 hours. Both the time and the dilution hinder the rapid correction of the coagulation defect.

Except for the provision of a highly purified and concentrated fibrinogen (my pending application, Serial No. 461,447 this is the first time that any coagulation components have been separated and made available in a form far `more useful clinically than the plasma from which they were derived. Furthermore, as pointed out in the description of clinical tests, the coagulation components of this invention are far more effective when administered simultaneously than when only one of the components is administered to correct a specific defect.

It should be pointed out that the product of this invention is devoid of thrombin, fibrinogen, thromboplastinogen (antihemophilia A factor) and accelerator globulin. Consequently, the product is of no value in the treatment of bleeding episodes due to deficiencies of fibrinogen, antihemophilia A factor (thromboplastinogen) or accelerator globulin.

I claim:

l. The method of collectively recovering lthe proteins: plasma thromboplastin component, proconvertin, and prothrombin from noncitrated human plasma, comprising: mixing said plasma with an insoluble barium salt, separating said adsorbing agent and its adsorbed proteins from the plasma, eluting the adsorbed proteins from said adsorbing agentv with a sodium citrate solution, adjusting the pH of the solution to a pH within the range of 3.0 to 4.5, precipitating the proteins with ethanol, and collecting the resulting precipitate.

2. The process of collectively precipitating a protein mixture of plasma thromboplastin component, proconvertin and prothrombin from a solution of proteins derived from noncitrated human plasma by adsorption on and elution from an insoluble barium salt, comprising: adjusting the pH of said solution to a pH within the range of 3.0 to 4.5 and adding a sufficient quantity of a lower aliphatic alcohol to precipitate said protein mixture.

3. The process of producing a mixture of plasma thromboplastin component, proconvertin and prothrombin from noncitrated human plasma comprising: contacting said plasma with insoluble barium salts; washing the contacted barium salts with normal saline solution; eluting the active proteins from the washed barium salts with sodium citrate solution; adjusting the pH of the eluate to a pH in the order of pH 7, to contain 15% ethanol, and to reduce the temperature to 5; separating the resulting supernatant solution and adjusting its pH to a value within the range of 3.2 to 4.5 and collecting the precipitate'. 1

References Cited in the tile of this patent Surgenor: J. Phy. and Colloid Chem., vol. 55, No. 1, pp. 94-101, January 1951.

Aggler: Science, vol. 119, No. 3101, June 4, 1954, pp. 806-807 (reprint).

Rimington: Biochem J., vol. 38, July 1944, pp. 54-60.

Cohn: J.A.C.S., vol. 72, January 1950, pp. 465-474 (esp. p. 466). 

1. THE METHOD OF COLLECTIVELY RECOVERING THE PROTEINS: PLASMA THROMBOPLASTIN COMPONENT, PROCONVERTIN, AND PROTHROMBIN FROM NON-CITRATED HUMAN PLASMA, COMPRISING: MIXING SAID PLASMA WITH AN INSOLUBLE BARIUM SALT, SEPARATING SAID ADSORBING AGENT AND ITS ADSORBED PROTEINS FROM THE PLASMA, ELUTING THE ADSORBED PROTEINS FROM SAID ADSORBING AGENT WITH A SODIUM CITRATE SOLUTION , ADJUSTING THE PH OF THE SOLUTION TO A PH WITHIN THE RANGE OF 3.0 TO 4.5, PRECIPITATING THE PROTEINS WITH ETHANOL, AND COLLECTING THE RESULTING PRECIPITATE. 